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At least 19 recordsLinked to original sources
PubMed2026

Proteomic analysis of ammonia-induced stress in Chinese hamster ovary (CHO) cell cultures.

Ammonia (NH₃) and its ionic form ammonium (NH₄+) are both metabolic waste products and essential nitrogen sources within Chinese hamster ovary (CHO) cell cultures. Although necessary for amino acid synthesis, excessive accumulation in the extracellular environment can exert stress, reducing cell proliferation and impairing the efficiency of recombinant protein production. Proper endoplasmic reticulum (ER) function is critical for CHO cells as biotherapeutic producers. Previous work has linked elevated ammonia concentrations to reduced productivity via altered N-glycosylation pathways, but its broader effects on ER biology remain unclear. In this study, we applied high-resolution mass spectrometry to perform a comprehensive analysis of changes in the ER proteome in CHO cells exposed to two ammonia concentrations, 10 mM and 30 mM, 48 and 120 h after supplementation. Both conditions suppressed cell growth and reduced product titre; however, the 10 mM supplementation resulted in a minor increase in specific cell productivity. Gene Ontology analysis revealed that ammonia strongly affected the tricarboxylic acid cycle, as well as key metabolic, catabolic and biogenetic processes. Several ER membrane proteins, including HMGCR and PREB, were consistently downregulated. In extended cultures, transmembrane proteins linked to Golgi-transport were upregulated, while vesicle transport associated proteins were downregulated, indicating altered intracellular trafficking. SIGNIFICANCE: This study provides a novel perspective on CHO cell biology under environmental stress by investigating the impact of ammonia accumulation in culture. Despite its presence in CHO culture, ammonia has been relatively under-investigated, compared to other culture conditions. Using high-throughput mass spectrometry for comprehensive proteomic profiling, we characterise the cellular response to ammonia build-up with a level of depth not previously applied to the study of this biological stressor. By specifically analysing proteins localised to the ER, we identify candidate pathways and molecular mechanisms that contribute to reduced CHO cell growth and productivity, offering insights directly relevant to industrial bioprocessing conditions. The link between ammonia concentration and a decrease in productivity has previously been linked to genes involved in N-glycosylation of the recombinant biotherapeutic, but the full extent of ammonia stress on ER function has not yet been investigated. These methods were applied to two IgG producing CHO cell lines to allow for comparison of cell line specific stress adaptations, as well as comparing the short- and long-term effects of excess ammonia.

Proteomics↗
PubMed2026

Schisantherin B mitigates cisplatin-induced ototoxicity by modulating the CNPY2-PERK/CHOP signaling axis.

Irreversible cisplatin-induced hearing loss (CIHL) is a refractory chemotherapy-related adverse effect with limited clinical treatments. Schisantherin B (STB), a lignan isolated from Schisandra chinensis, is widely recognized for its neuroprotective properties, while its role in auditory injury remains unclear. Herein, we found that STB alleviated cisplatin-induced ototoxicity in House Ear Institute Organ of Corti 1 (HEI-OC1) cells and guinea pig models, protecting cochlear hair cells, synaptic ribbons and spiral ganglion neurons, and partially restoring auditory brainstem response (ABR) thresholds. Furthermore, combined drug affinity responsive target stability (DARTS) assay, the cellular thermal shift assay (CETSA), and the surface plasmon resonance (SPR) assay, we confirmed STB directly binds to the canopy FGF signaling regulator 2 (CNPY2), a key initiator of endoplasmic reticulum (ER) stress. Notably, consistent dual in vitro and in vivo validation confirmed that STB exerts no regulatory effect on CNPY2 protein abundance, yet suppressed the downstream Protein kinase R-like endoplasmic reticulum kinase / C/EBP homologous protein (PERK/CHOP) signaling cascade and ER stress-mediated apoptosis. Moreover, molecular docking and co-immunoprecipitation (co-IP) validated the physical binding of STB to CNPY2 and the endogenous interaction between CNPY2 and PERK. Additionally, CNPY2 overexpression and shRNA knockdown further verified this functional relationship. Integrated proteomic and transcriptomic analyses showed STB partially reversed cisplatin-triggered inflammation and excessive ER stress. Collectively, our results suggest STB may serve as a potential otoprotective agent. The CNPY2-PERK/CHOP axis is closely linked to cisplatin-induced cochlear damage and offers a feasible target for intervention against CIHL. Abbreviations: CIHL, cisplatin-induced hearing loss; STB, Schisantherin B; HEI-OC1, house ear institute organ of corti 1; ABR, auditory brainstem response; DARTS, drug affinity responsive target stability; CETSA, cellular thermal shift assay; SPR, surface plasmon resonance; CNPY2, canopy FGF signaling regulator 2; ER, endoplasmic reticulum; PERK, protein kinase R-like endoplasmic reticulum kinase; CHOP, C/EBP homologous protein; co-IP, co-immunoprecipitation; STA, Schisantherin A; STC, Schisantherin C; dB SPL, decibels sound pressure level; EDTA, ethylenediaminetetraacetic acid; dB SPL, decibels sound pressure level; SGN, spiral ganglion neuron; IHCs, inner hair cells; OHCs, outer hair cells; CCK-8, Cell Counting Kit-8; OD, optical density; ODb, blank sample, ODc, control sample; NC, negative control; PVDF, polyvinylidene difluoride; RT, room temperature; LC-MS/MS, liquid chromatography tandem mass spectrometry; MS, mass spectrometry; DMSO, dimethyl sulfoxide; KDs, equilibrium dissociation constants; SP, standard precision; SEM, standard error of the mean; HSD, honestly significant difference; Ctrl, control group; CV, cell viability; Kd, dissociation rate constant; Ka, association rate constant; STS, sodium thiosulfate; UPR, unfolded protein response; BLB, blood-labyrinth barrier.

Apoptosis↗
PubMed1976

Temperature dependent changes in the thyroid gland of Mertensiella caucasica (Urodela, Amphibia).

The influence of temperature on the thyroid gland of the Caucasian salamander, Mertensiella caucasica has been studied (1) with the light- and electron microscopes, (2) with enzyme histochemical and quantitative methods. Animals which had been kept for years at 16 degrees C were exposed for 48 hrs to 4 degrees C or 27 degrees C. At 4 degrees C the follicular epithelium consisted of cuboidal cells with well-developed rough ER, occasional distended cisternae of the rough endoplasmic reticulum (ER) filling most of the cytoplasm, a relatively small Golgi apparatus and numerous round or oval shaped lysosomes. In animals kept at 27 degrees C the follicular wall was composed of markedly taller epithelial cells, the apices of which bulged into the follicular lumen. Compared to the animals at 4 degrees C, there appeared to be a decrease in the rough ER. The Golgi apparatus did not exhibit clear differences. The lysosomes increased in number and showed various shapes among which elongated ones with pointed ends were particularly striking. In the apical cytoplasm colloid droplets appeared indicating hormone release. The apical microvilli increased in number. The measurements of total T4 have shown that the hormone level in animals kept at 27 degrees C was about twice as high as in animals kept at 4 degrees C.

Animals↗
PubMed1978

Annulate lamellae in a malignant mesenchymal tumor.

Intracytoplasmic membranous structures, known as annulate lamellae were seen in a malignant mesenchymal tumor. Close relationship with rough endoplasmic reticulum (ER) suggested their origin from ER.

Cytoplasm↗
PubMed2025

Convergent activation of the integrated stress response and ER-mitochondria uncoupling in VAPB-associated ALS.

Vesicle-associated membrane protein-associated protein-B (VAPB) is an endoplasmic reticulum (ER) membrane-bound protein. The P56S mutation in VAPB causes a dominant, familial form of amyotrophic lateral sclerosis (ALS). However, the mechanism by which this mutation leads to motor neuron (MN) degeneration remains unclear. Utilizing inducible pluripotent stem cell (iPSC)-derived MNs expressing either wild-type (WT) or P56S VAPB, we demonstrate that the mutant protein reduces neuronal firing and disrupts ER-mitochondria-associated membranes (ER MAMs), with a time-dependent decline in mitochondrial membrane potential (MMP), hallmarks of MN pathology. These findings were validated in patient-derived iPSC-MNs. Additionally, VAPB P56S MNs show increased susceptibility to ER stress, elevated expression of the Integrated Stress Response (ISR) regulator ATF4 under stress, and reduced global protein synthesis. Notably, pharmacological ISR inhibition using ISRIB rescued ALS-associated phenotypes in both VAPB P56S and patient-derived iPSC-MNs. We present the first evidence that the VAPB P56S mutation activates ISR signaling via mitochondrial dysfunction in human MNs. These findings support ISR modulation as a strategy for ALS intervention and highlight the need for patient stratification in clinical trials.

Amyotrophic Lateral Sclerosis↗
PubMed1976

The beads in the Golgi complex-endoplasmic reticulum region.

The region between the rough endoplasmic reticulum (ER) and the Golgi complex has been studied in a variety of insect cell types in an attempt to find a marker for the exit gate or gates from the ER. We have found that the smooth surface of the rough endoplasmic reticulum near Golgi complex transitional elements has beadlike structures arranged in rings at the base of transition vesicles. They occur in all insect cell types and a variety of other organisms. The beads can be seen only after staining in bismuth salts. They are 10-12 nm in diameter and are separated from the membrane and one another by a clear halo giving them a center to center spacing of about 27 nm. The beads are not sensitive to nucleases under conditions which disrupt ribosomes or remove all Feulgen staining material from the nucleus. Under conditions similar to those used to stain tissue, bismuth does not react in vitro with nucleic acids. The component of the beads that stains preferentially with bismuth is therefore probably not nucleic acid.

Bismuth↗
PubMed1978

Unusual configurations of endoplasmic reticulum in cells of acute promyelocytic leukemia.

An ultrastructural study of leukemia cells from 8 patients with acute promyelocytic leukemia revealed several features that have not previously been emphasized: prominent dilated rough endoplasmic reticulum and two unusual configurations of endoplasmic reticulum (ER). The two membrane structures, multilaminar ER and complex stellate arrangements of ER, appeared to be morphogenetically related. The multilaminar ER was observed in every mitotic cell and less frequently in interphase cells. The stellate ER complex was observed only in interphase cells. Ultrastructural evidence is presented to support the possible evolution of the stellate ER complex from the multilaminar ER.

Endoplasmic Reticulum↗
PubMed1978

Spatial orientation of glycoproteins in membranes of rat liver rough microsomes. I. Localization of lectin-binding sites in microsomal membranes.

Carbohydrate-containing structures in rat liver rough microsomes (RM) were localized and characterized using iodinated lectins of defined specificity. Binding of [125I]Con A increased six- to sevenfold in the presence of low DOC (0.04--0.05%) which opens the vesicles and allows the penetration of the lectins. On the other hand, binding of [125I]WGA and [125I]RCA increased only slightly when the microsomal vesicles were opened by DOC. Sites available in the intact microsomal fraction had an affinity for [125I]Con A 14 times higher than sites for lectin binding which were exposed by the detergent treatment. Lectin-binding sites in RM were also localized electron microscopically with lectins covalently bound to biotin, which, in turn, were visualized after their reaction with ferritin-avidin (F-Av) markers. Using this method, it was demonstrated that in untreated RM samples, binding sites for lectins are not present on the cytoplasmic face of the microsomal vesicles, even after removal of ribosomes by treatment with high salt buffer and puromycin, but are located on smooth membranes which contaminate the rough microsomal fraction. Combining this technique with procedures which render the interior of the microsomal vesicles accessible to lectins and remove luminal proteins, it was found that RM membranes contain binding sites for Con A and for Lens culinaris agglutinin (LCA) located exclusively on the cisternal face of the membrane. No sites for WGA, RCA, soybean (SBA) and Lotus tetragonobulus (LTA) agglutinins were detected on either the cytoplasmic or the luminal faces of the rough microsomes. These observations demonstrate that: (a) sugar moieties of microsomal glycoproteins are exposed only on the luminal surface of the membranes and (b) microsomal membrane glycoproteins have incomplete carbohydrate chains without the characteristic terminal trisaccharides N-acetylglucosamine comes from galactose comes from sialic acid or fucose present in most glycoproteins secreted by the liver. The orientation and composition of the carbohydrate chains in microsomal glycoproteins indicate that the passage of these glycoproteins through the Golgi apparatus, followed by their return to the endoplasmic reticulum, is not required for their biogenesis and insertion into the endoplasmic reticulum (ER) membrane.

Animals↗
PubMed2026

The Sigma1 ER membrane receptor promotes structural protein folding and genome packaging of dengue virus.

Dengue virus (DENV) exploits the host endoplasmic reticulum (ER) to support viral protein translation and folding, replication, and assembly, although the identity of ER factors that promote these distinct steps during infection remain unclear. Here we demonstrate that the ER-resident Sigma1 ER membrane receptor (S1R) promotes virus structural protein folding and genome packaging of DENV during infection. Under S1R knockdown (KD), DENV infection is impaired without compromising virus translation or replication. Strikingly, EM analysis revealed that DENV particles in and secreted from S1R-depleted cells are smaller, likely because they are empty particles devoid of the vRNA genome. Biochemical experiments demonstrated that S1R binds to the prM structural protein and under S1R KD, the prM, E and C structural proteins became detergent-insoluble. Thus, without S1R, all three virus structural proteins misfold, impairing efficient genome packaging. Together, these findings identify a novel ER chaperone that supports a critical DENV infection step.

Dengue Virus↗
PubMed1979

Differences in the incorporation of [3H]-glucosamine into nascent polypeptide chains on free polysomes and two fractions of membrane-bound polysomes in mouse myeloma cells.

The incorporation of [3H]-glucosamine into polypeptides of three fractions of polysomes in MPC-11 cells was studied. After short term incubation greatest incorporation was observed in a fraction of membrane-bound polysomes, which after nitrogen cavitation of cells, remained bound to the endoplasmic reticulum (ER) associated with the nucleus (fraction 2). Polypeptide chains on membrane-bound polysomes in the microsomal fraction (fraction 1) and free polysomes contained much less radioactivity. Since nascent polypeptide chains contained within membrane-bound polysomes of fraction 2 are glycosylated at an earlier stage than those in fraction 1 it is likely that this represents a difference in type of proteins synthesized in the respective fractions of ER.

Cell Line↗
PubMed1977

Alterations in the endoplasmic reticulum and Golgi complex of intestinal epithelial cells during fat absorption and after termination of this process: a morphological and morphometric study.

Earlier investigations of intestinal fat-absorption have stressed the importance of continued protein synthesis to provide membranes which are utilized for the intracellular transport of resynthesized lipid. The resulting membranes, when incorporated into the endoplasmic reticulum (ER) and Golgi complex, serve as vehicles for the movement of fat within the cell and for its release to the extracellular space. In the current study, attention was focused on the morphological changes in the ER and Golgi complex both during fat absorption and at successive time intervals after fat-absorption termination. Morphological interpretations were confirmed by morphometric analysis. This investigation supports the interpretation that during fat absorption, membrane synthesis by the rough endoplasmic reticulum (RER) is insufficient to accomodate membrane utilization and intraconversion, resulting in a decrease of both ER and Golgi complex components. However, following fat-absorption termination, and cell is able to replace previously depleted components of the ER and Golgi complex and regain the full membrane complement of the fasted state. Replenishment of cellular membranes is postulated as resulting from a continued synthesis of new membranes by the RER which eventually exceeds membrane utilized during lipid transport.

Animals↗
PubMed1976

Prevention by steroids of cerium hepatotoxicity.

In female rats, the lethality and hepatotoxicity of cerous chloride (CeCl3) were significantly altered by pretreatment with steroidal and nonsteroidal compounds (pregnenolone-16alpha-carbonitrile "PCN", dexamethasone, spironolactone, phenobarbital) that stimulate hepatic drug-metabolizine enzyme activity and by estradiol. PCN, estradiol, and dexamethasone considerably decreased hepatic triglycerides, but the latter steroid also greatly sensitized the animals to CeCl3 lethality. Spironolactone and phenobarbital similarly lowered the triglyceride level but not significantly. Light and electron microscopy indicated that the hepatocytic damage elicited by CeCl3 was decreased significantly by PCN, estradiol, and dexamethasone. The steroids may have altered the distribution and binding of the metal to the endoplasmic reticulum (er) thus protecting these membranes against the effect of cerium.

Animals↗
PubMed1979

Retinal pigment epithelium. Interrelations of endoplasmic reticulum and melanolysosomes in the black mouse and its beige mutant.

By cytochemistry (acid phosphatase and tyrosinase activities) GERL, a specialized hydrolase-rich region of endoplasmic reticulum (ER), can be visualized in the cells of the mouse retinal pigment epithelium. Previously catalase cytochemistry permitted us to identify microperoxisomes, with numerous continuities to the ER. The present report reveals the extensive continuities of the ER to pigment granules in various stages of maturation. When the pigment granules, which we consider to be "melanolysosomes," first appear they consist of electron-opaque grains within dilated areas of the ER. As the dilations enlarge, fine fibrils appear in the ER cisternae. Thicker fibers develop from the fibrils; these fibers are generally obscured when melanin deposition occurs. At all stages, the melanolysosomes appear to be connected to the ER.

Acid Phosphatase↗
PubMed2026

Intermembrane coupling between Bcl-xL and the IP3 receptor supports local Ca2+ transfer at ER-mitochondrial contacts.

Bcl-xL, an anti-apoptotic Bcl-2 family protein, engages laterally with Bak/Bax in the outer mitochondrial membrane (OMM) to inhibit apoptosis and interacts with the IP3 receptor Ca2+ channels (IP3Rs) in the endoplasmic reticulum (ER) membrane to control Ca2+ release. It is unknown if OMM-localized Bcl-xL can also interact in trans with IP3Rs at ER-mitochondrial contacts to form a tethering complex that supports IP3R-mediated local Ca2+ transfer from ER to mitochondria. We establish that IP3R-mitochondria Ca2+ signal propagation depends on Bcl-xL. By targeting Bcl-xL specifically to different subcellular compartments, we find that OMM-localized Bcl-xL increases the efficacy of ER-mitochondrial Ca2+ transfer without changing ER Ca2+ release, despite attenuating mitochondrial Ca2+ uptake. We find interaction between Bcl-xL and each IP3R isoform occurring at the mitochondria and a complex formed by OMM-localized Bcl-xL and IP3Rs. OMM Bcl-xL interacts with IP3Rs in trans at ER-mitochondrial contacts to optimize local Ca2+ signal propagation into the mitochondria.

Bcl-xL↗
PubMed2025

Establishment of a prognostic model based on ER stress-related cell death genes and proposing a novel combination therapy in acute myeloid leukemia.

BACKGROUND: Acute myeloid leukemia (AML) is a highly heterogeneous malignancy, presenting significant challenges in accurately predicting patient prognosis. Dysregulation of endoplasmic reticulum (ER) stress and resistance to programmed cell death (PCD) are hallmarks of AML cells. However, the prognostic significance of the interplay between ER stress and cell death pathways in AML remains largely unexplored. METHODS: We analyzed RNA sequencing and clinical data from 887 AML patients across 4 cohorts to develop an ER stress-related cell death index (ERCDI) using 10 machine-learning algorithms with 117 unique combinations. Survival and time-dependent Receiver Operating Characteristic Curve (ROC) analyses were performed to assess the model's efficacy. Clinical characteristics, the tumor immune microenvironment, and drug sensitivity differences between the high- and low-risk groups were also analyzed. The CMap database was used to identify potential therapeutic drugs. In vitro and in vivo experiments, including CCK-8, colony formation, flow cytometry, Transwell assays, and xenograft mouse models, were conducted to evaluate the effects of the target genes and candidate drugs. RESULTS: The ERCDI demonstrated strong prognostic and predictive performance for prognosis in AML patients. Furthermore, the ERCDI effectively predicted immunotherapy and chemotherapy outcomes and was associated with the immune features of the different risk groups. DNA damage-inducible transcript 4 protein (DDIT4), a key gene associated with ERCDI, is related to poor prognosis in AML patients with high expression. Additionally, the knockdown of DDIT4 significantly inhibited AML cell proliferation, induced cell apoptosis, and promoted cell cycle arrest. Chaetocin was subsequently identified as a candidate compound for AML treatment. Subsequent experiments suggested that combining chaetocin and venetoclax is a potentially promising therapeutic strategy for AML. CONCLUSION: The ERCDI provides personalized risk assessment and treatment recommendations for individual AML patients. The combined use of chaetocin and venetoclax can potentially be repurposed for AML therapy.

Humans↗
PubMed1976

Proliferation of endoplasmic reticulum with its enzyme, UDP-glucuronyltransferase, in chick embryo liver during culture. Effects of phenobarbital.

UDP-Glucuronyltransferase (GT) activity increases in chick embryo liver during culture from zero to a steady-state level at or above adult values. The GT activity (o-aminophenol as acceptor) is located entirely in the membranes of the endoplasmic reticulum (ER) and the question arises whether ER increases along with GT. Earlier work showed that the synthesis and degradation rates of GT can be varied in culture over wide ranges by choosing embryo livers of different ages and both phenobarbital. In the present study we measured the GT activities and the concentrations of ER (using stereologic methods) in 5- and 11-day embryo liver during culture with and without phenobarbital. We found that GT and ER always increased in a constant ratio of 2.2 X 10(-9) U of GR activity per square micrometer of membrane, suggesting that the synthesis and degradation of GT are coupled to the synthesis and degradation of ER. A general structure for ER is proposed to explain this finding.

Animals↗
PubMed2026

Reprogrammed Komagataella phaffii for enhanced secretory expression of human lactoferrin.

Human lactoferrin (hLF) is a multifunctional glycoprotein of the transferrin family derived from milk and mucosal secretions, which exhibits antibacterial, anti-tumor, and immunomodulatory functions, and is an important component of infant formula. Conventional methods for lactoferrin expression are often inefficient, primarily due to inadequate protein synthesis capabilities and poor stability within microbial hosts. Herein, a Komagataella phaffii yeast strain capable of high-level secretory expression of hLF was constructed by reprogramming the endoplasmic reticulum (ER) and vacuole using CRISPR/Cas9 technology. A dual-expression cassette containing the AOX1 promoter, an α-secretion signal peptide, the hLF gene, and a terminator was integrated into three different sites of the K. phaffii genome. The stepwise strategy combining expansion of the ER membrane involved in protein synthesis with knockout of vacuolar proteases further enhanced hLF production. Subsequently, 0.1 g/L FeCl₃ was added to the medium to reduce the toxicity of hLF and improve its stability. After high-density cultivation of K. phaffii through optimization of cultivation conditions in shake flasks and a 5 L bioreactor, the secretory intact hLF titer reached 2214 mg/L, representing a 76.3-fold increase achieved through these engineering strategies. In addition, antibacterial experiments demonstrated that this secretory hLF had a significant inhibitory effect on Escherichia coli, Staphylococcus aureus, and yeast. Overall, the developed K. phaffii protein expression platform enabled efficient production of lactoferrin, demonstrating its potential for expressing other lactoproteins.

Lactoferrin↗
PubMed1977

Microtubules associated with postsynaptic 'thickenings'.

Using a new albumin technique, microtubules can be seen closely related to or associated with the postsynaptic 'thickening' of mature and immature central nervous synapses. Thus in conventionally fixed synapses (without albumin pretreatment) where microtubules cannot usually be observed running into the postsynaptic dense material, this material could perhaps, in part, consist of the debris of in vivo microtubules. Smooth endoplasmic reticulum (ER) is often seen associated with the microtubules near the postsynaptic 'thickening'. Microtubules, and possibly smooth ER, may have an important role in the initiation of synapse formation and in the maintenance of mature synapses.

Age Factors↗